论文部分内容阅读
目的观察BC047440基因能否通过增强NF-κB的活化而促进HepG2细胞的增殖。方法设计针对BC047440基因的小干扰RNA(siRNA),构建pGenesil-1-BC047440-siRNA真核表达载体,用脂质体法将介导siRNA的真核绿色荧光表达载体pGenesil-1-BC047440-siRNA、pGenesil-1-HK-siRNA转染人肝癌HepG2细胞建立稳定细胞株;实验分为3组:转染siRNA表达载体组、无关质粒组和未处理的亲本HepG2细胞组。采用平板克隆形成实验分析细胞的增殖活性,通过Western blot检测细胞质p50含量,EMSA检测NF-κB的核转位,荧光素酶试验检测各组细胞的NF-κB活化情况。结果细胞增殖实验显示转染siRNA组细胞增殖能力明显降低,BC047440基因沉默后的HepG2的细胞质p50含量低于野生株HepG2,NF-κB核转位低于野生株HepG2,其荧光素酶和海肾荧光素酶活性比值只有野生株的1/4。结论BC047440基因能促进肝癌细胞的增殖,其调节机制可通过NF-κB信号途径实现,提示BC047440基因可能成为抑制人肝癌细胞生长的有效靶点。
Objective To observe whether BC047440 can promote the proliferation of HepG2 cells by enhancing the activation of NF-κB. Methods The eukaryotic expression vector pGenesil-1-BC047440-siRNA targeting BC047440 gene was designed and its eukaryotic expression vector pGenesil-1-BC047440-siRNA was constructed by liposome method. pGenesil-1-HK-siRNA was transfected into HepG2 cells to establish a stable cell line. The experiment was divided into 3 groups: transfected siRNA expression vector group, unrelated plasmid group and untreated HepG2 cells. Cell proliferation was analyzed by plate clone formation assay. The p50 content of cytoplasm was detected by Western blot. The nuclear translocation of NF-κB was detected by EMSA. The activation of NF-κB was detected by luciferase assay. Results The results of cell proliferation assay showed that the proliferation of HepG2 cells transfected with siRNA was significantly lower than that of HepG2 cells transfected with BC047440 gene. The nuclear translocation of NF-κB in HepG2 cells was lower than that in wild type HepG2 cells. The luciferase and Renilla The luciferase activity ratio was only 1/4 of the wild-type strain. CONCLUSION: BC047440 gene can promote the proliferation of hepatocellular carcinoma cells. The regulatory mechanism may be through NF-κB signaling pathway, suggesting that BC047440 gene may be an effective target for inhibiting the growth of human hepatocellular carcinoma cells.